RESUMO
Gelatinases play important roles in tumour invasion and metastasis and are thus considered promising targets for cancer therapy. In this study, a new single-chain variable fragment (scFv)-based fusion protein Fv-LDP, composed of the anti-gelatinases scFv and lidamycin apoprotein (LDP), was prepared, and its combination with angiogenesis inhibitor Endostar was then investigated. The fusion protein Fv-LDP specifically bound to various tumour cells, and its binding capability to human pulmonary giant cell carcinoma (PG) cells was higher than that of LDP. Fv-LDP inhibited the expression and secretion of gelatinases and could be internalized into tumour cells via endocytosis. Fv-LDP also suppressed the growth of human hepatoma cells and murine hepatoma 22 transplanted in Kunming mice in various degrees. In addition, Endostar could enhance the synergistic or additive inhibition of Fv-LDP on the growth, migration or invasion of human hepatoma cells shown by a colony formation assay and a transwell-based migration or invasion assay, respectively. In vivo, Fv-LDP/Endostar combination showed a significantly synergistic effect on the growth of a human hepatoma xenograft, with an inhibition rate of 80.8% compared with the Fv-LDP (44.1%) or Endostar (8.9%)-treated group. The above-mentioned results indicate that the fusion protein Fv-LDP is effective against transplantable hepatoma in mice and human hepatoma xenografts in athymic mice. Moreover, Endostar can potentiate the inhibition effect of Fv-LDP on the growth of human hepatoma cells and xenografts. These data will provide a new combined strategy for improving the therapeutic efficacy of treatments for hepatoma or other gelatinase-overexpressing tumours.
Assuntos
Aminoglicosídeos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Endostatinas/farmacologia , Enedi-Inos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Anticorpos de Cadeia Única/farmacologia , Aminoglicosídeos/administração & dosagem , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacologia , Animais , Apoproteínas/administração & dosagem , Apoproteínas/farmacologia , Carcinoma de Células Gigantes/tratamento farmacológico , Carcinoma de Células Gigantes/enzimologia , Carcinoma de Células Gigantes/patologia , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Endostatinas/administração & dosagem , Enedi-Inos/administração & dosagem , Feminino , Gelatinases/metabolismo , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Recombinantes , Anticorpos de Cadeia Única/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have chemically synthesized NGR-LDP-PYM, a novel CD13/aminopeptidase (APN)-targeting and hydrolase-resistant conjugate by cross-linking of the antitumor antibiotic pingyangmycin (bleomycin A5 , PYM) to an engineered NGR motif-integrated apoprotein (NGR-LDP) with a noncleavable linker. This protein-drug conjugate not only basically retains the original properties of PYM but also can specifically deliver PYM to the CD13/APN-expressing tumor cells. Furthermore, the resulting conjugate exhibits more resistance to hydrolysis of recombinant human bleomycin hydrolase than parental PYM. These results may be useful for improving the therapeutic efficacy of PYM and have implications in the treatment of PYM-refractory and CD13/APN-overexpressing tumors.
Assuntos
Aminopeptidases/metabolismo , Antibióticos Antineoplásicos/administração & dosagem , Apoproteínas/química , Bleomicina/análogos & derivados , Antígenos CD13/metabolismo , Sistemas de Liberação de Medicamentos , Sequência de Aminoácidos , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Anticorpos Monoclonais , Apoproteínas/metabolismo , Bleomicina/administração & dosagem , Bleomicina/química , Bleomicina/metabolismo , Linhagem Celular Tumoral , Humanos , Hidrolases/metabolismo , Hidrólise , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
LPS stimulation of macrophages production of IFN-beta plays a key role in innate immunity defending the microbial invasion. In this study, the effect of S632A3 promoting LPS-induced IFN-beta production and the underlying mechanism were investigated, mRNA level was measured by real-time PCR, cytokine production was determined by ELISA, GSK-3beta activity was investigated by kinase assay, protein phosphorylation and expression were evaluated by Western blotting. The results revealed that S632A3 significantly augmented IFN-beta production by LPS-stimulated macrophages. S632A3 inhibition of the activation of GSK-3beta, reduced the threonine 239 phosphorylation of transcription factor c-Jun but increased the total level of c-Jun in LPS-stimulated macrophages. Moreover, small interfering RNA-mediated knockdown of c-Jun level abrogated the ability of S632A3 to augment IFN-beta. The study thus demonstrates S632A3 being a new anti-inflammation lead compound and provides a molecular mechanism by which S632A3 promoted LPS-induced IFN-beta production in macrophages through inhibiting the activation of GSK-3beta.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Interferon beta/biossíntese , Macrófagos/metabolismo , Piperidonas/farmacologia , Animais , Antibacterianos/farmacologia , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta , Interferon beta/genética , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
This study is to optimize the preparation process of fusion protein Fv-LDP which was expressed in the form of inclusion body and consisted of lidamycin apoprotein LDP and single-chain Fv antibody (scFv) directed against type IV collagenase. The preparation and the dissolution of inclusion body, the immobilized metal affinity chromatography of the target protein and the renaturization by stepwise dialysis were optimized by single-factor analysis or orthogonal design. In addition, the refolded fusion protein Fv-LDP was refined by Sephadex G-75 chromatography followed by fluorescence-activated cell sorter (FACS)-based saturation binding assay to measure its antigen-binding activity. After optimization of the process, the purity of fusion protein Fv-LDP existed in the inclusion body was 63.9% and the corresponding solubility was 95.7%; Under denaturing conditions, the purity of fusion protein Fv-LDP was more than 95% after the purification process. The percentage of monomeric fusion protein Fv-LDP was 60% after the refolding process, while it was further refined to 85% which was 5.6-fold higher than that of the initial refolding condition. The refined fusion protein Fv-LDP could bind to human lung adenocarcinoma PAa cells and human hepatoma BEL-7402 cells with the dissociation constants (Kd) of 0.176 micromol x L(-1) and 0.904 micromol x L(-1), respectively. The preparation process of fusion protein Fv-LDP has been successfully optimized, which provides the experimental basis for the production and future development of fusion protein Fv-LDP, and might serve as a relatively practical system for the preparation of other scFv-based proteins expressed in the form of inclusion body.
Assuntos
Aminoglicosídeos , Apoproteínas , Enedi-Inos , Proteínas Recombinantes de Fusão , Anticorpos de Cadeia Única , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Colagenases/imunologia , Enedi-Inos/química , Enedi-Inos/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/metabolismoRESUMO
Inflammatory mediators including inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) contribute to the course of a variety of inflammatory diseases. S632A3 is a new member of the glutarimide antibiotics isolated from a cultured broth of Streptomyces hygroscopicus S632 with a potent NF-κB inhibitory activity. In the present study, we investigated the anti-inflammatory effects and the underlying molecular mechanism of S632A3 on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. S632A3 concentration-dependently inhibited LPS-induced NO and prostaglandin E(2) (PGE(2)) production through the suppression of iNOS and COX-2 at gene transcription levels. In addition, S632A3 suppressed NF-κB-dependent inflammatory responses by inhibiting the activation of glycogen synthase kinase 3ß (GSK-3ß), while the activation of IκB kinase (IKK) complex was unaffected. S632A3 suppressed NF-κB activity by differentially affecting the CREB (cAMP response element-binding protein) and NF-κB p65 interacting with the coactivator CBP (CREB binding protein). S632A3 also inhibited GSK-3ß-elicited iNOS and COX-2 expression. Moreover, S632A3 was shown to inhibit the activation of ASK1 (Apoptosis-signal regulating kinase 1) and p38 mitogen-activated protein kinase, therefore attenuated the LPS-induced NF-κB activity in macrophages. Furthermore, S632A3 significantly reduced the pro-inflammatory cytokines TNF-α and IL-6 production while increased the anti-inflammatory cytokine IL-10 production in LPS-stimulated RAW264.7 cells. Our study thus provides a molecular mechanism by which S632A3 inhibited LPS-induced pro-inflammatory response in macrophages through interfering with the activation of GSK-3ß and ASK1-p38 signaling.
Assuntos
Antibacterianos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Piperidonas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular , Glicogênio Sintase Quinase 3 beta , Mediadores da Inflamação/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/patologia , CamundongosRESUMO
Na(+)/K(+)-ATPase targeted cancer therapy has attracted increasing interests of oncologists in lung cancer field. Although multiple anti-cancer mechanisms of cardiac glycosides as Na(+)/K(+)-ATPase inhibitors are revealed, the role of autophagy and related molecular signaling pathway for the class of compounds in human non-small cell lung cancer (NSCLC) cells has not been systematically examined. We herein investigated the anti-cancer effects of two representative cardiac glycosides, digoxin and ouabain, in A549 and H460 cell lines. Both agents caused significant growth inhibition at nanomolar level. The cardiac glycosides were found to induce moderate G(2)/M arrest but not apoptosis at IC(50) level in the NSCLC cell lines. Moreover, autophagy was markedly induced by both agents, as evidenced by the time- and dose-dependent increase of LC3-II, up-regulation of Atg5 and Beclin1, as well as by the observations through acridine orange staining, transmission electron microscopy and quantification of GFP-LC3 fluorescence. Importantly, AMP-activated protein kinase (AMPK) pathway was activated, resulting in mammalian target of rapamycin (mTOR) deactivation during autophagy induction. Moreover, extracellular-signal-regulated kinase 1/2 (ERK1/2) activation was simultaneously found to be involved in the autophagy regulation. Co-treatment with respective inhibitors or siRNAs could either block the autophagic phenotypes and signals, or significantly increase the cellular viability, indicating the drugs-induced autophagy plays tumor-suppressing role. This work provides first evidence showing that the cardiac glycosides induce autophagy in human NSCLC cells through regulation of both mTOR and ERK1/2 signaling pathways. The autophagy may at least partially account for the growth inhibitory effects of the compounds in human NSCLC cells.
Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Glicosídeos Cardíacos/farmacologia , Neoplasias Pulmonares/patologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Androstadienos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Biomarcadores/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Linhagem Celular Tumoral , Digoxina/farmacologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/ultraestrutura , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Ouabaína/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura , WortmaninaRESUMO
The glycoprotein erythropoietin (Epo) is a hematopoietic cytokine necessary for the survival of erythrocytes from immature erythroid cells. The mitogen-activated c-Jun N-terminal kinase 1 (JNK1) plays an important role in the proliferation and survival of erythroid cells in response to Epo. However, the precise mechanism of JNK1 activation promoting erythroid cell survival is incompletely understood. Here, we reported that JNK1 is required for Epo-mediated cell survival through phosphorylation and inactivation of the pro-apoptotic, Bcl-2 homology domain 3 (BH3)-only Bcl-associated death protein (Bad). Upon Epo withdrawal, HCD57 cells, a murine Epo-dependent cell line, displayed increased apoptotic cell death that was associated with decreased JNK1 activity. Epo withdrawal-induced apoptosis was promoted by inhibition of JNK1 activity but suppressed by expression of a constitutively active JNK1. Furthermore, Epo-activated JNK1 phosphorylated Bad at threonine 201, thereby inhibiting the association of Bad with the anti-apoptotic molecule B-cell lymphoma-extra large (Bcl-X(L)). Replacement of threonine 201 by alanine in Bad promoted Epo withdrawal-induced apoptosis. Thus, our results provide a molecular mechanism by which JNK1 contributes to the survival of erythroid cells.
Assuntos
Eritropoetina/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismoRESUMO
Chemomicin (CHM), an angucyclinone antibiotic extracted from the fermentation broth of Nocardia Mediterranei subsp. Kanglensis 1747-64, shows immunosuppressive activity. However, whether it can inhibit growth of tumor cells remains elusive. In the present study, we show that CHM potently inhibited the proliferations of eight various types of human tumor cell lines and non-cross resistant to multidrug-resistant cells. In contrast to action of doxorubicin, the generation of reactive oxygen species was observed as early as 30 min after addition of CHM and its process did not involve iron. The apoptotic cells with chromatin condensation and Annexin V staining markedly increased after the human hepatoma HepG2 was exposed to 1, or 2 microg/ml CHM for 24 h. In the CHM-induced apoptosis, robust increment of p53 expression, activation of caspase-3, -7, -8, -9, cleavage of PARP and the phosphorylation of p38 and JNK, were detected by Western blot analysis. Further investigation revealed the disruption of mitochondrial membrane potential in the cells with CHM incubation for 4 h. Taken together, the results demonstrated that potent proliferation inhibitory effect of CHM on tumor cells is due to activation of the apoptotic pathway.
Assuntos
Antraquinonas/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias/patologia , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Fase G2/efeitos dos fármacos , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
OBJECTIVE: Lidamycin, an enediyne antibiotic, leads to apoptosis and mitotic cell death of human tumor cells at high and low concentrations. The reason why tumor cells have distinct responses to lidamycin remains elusive. This study was to elucidate if cellular prosurvival molecules are involved in these responses. METHODS: Cleavage of chromatin and DNA was observed by chromatin condensation and agarose gel electrophoresis. Accumulation of rhodamine 123 in lidamycin-treated cells was assayed by flow cytometry. Cell multinucleation was detected by staining with Hoechst 33342. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively. RESULTS: SIRT1 deacetylase remained unchanged in 0.5 nmol/L lidamycin whereas cleavage occurred when apoptosis was induced by lidamycin. Increased FOXO3a, SOD-1 and SOD-2 expression and transient phosphorylation of ERK were detected after exposure of human hepatoma BEL-7402 cells to 0.5 nmol/L lidamycin. High expressions of SIRT1 and Akt were found in colon carcinoma HCT116 p53 knock-out cells exposed to lidamycin. Degradation of PARP and p53 by lidamycin as a substitute for SIRT1 and Akt was confirmed with caspase inhibitor Q-VD-OPh and proteasome inhibitor MG132. Resistance to lidamycin-induced DNA cleavage was observed in breast cancer doxorubicin-resistant MCF-7 cells. This was not induced by P-glycoprotein as no accumulation of rhodamine 123 was detected in the resistant cells following exposure to lidamycin. In contrast to sensitive MCF-7 cells, a lower multinucleation rate for the resistant cells was measured following exposure to equal concentrations of lidamycin. CONCLUSIONS: Cellular prosurvival molecules, such as SIRT1, Akt, SOD-1, SOD-2 and other unknown factors can influence the action of lidamycin on human tumor cells.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Enedi-Inos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Clivagem do DNA , Doxorrubicina/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sirtuína 1 , Sirtuínas/genética , Sirtuínas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVE: To study whether Lycium barbarum glycopeptide 3 (LBGP3) affects T cell apoptosis in aged mice. METHODS: LBGP3 was purified with DEAE cellulose and Sephadex columns. Apoptotic "sub-G1 peak" was detected by flow cytometry and DNA ladder was resolved by agarose gel electrophoresis. Levels of IFN-gamma and IL-10 were measured with specific kits and mRNA expression was detected by RT-PCR. Apoptosis-related proteins of FLIP, FasL, and Bcl-2 were determined by Western blotting. RESULTS: LBGP3 was purified from Fructus Lycii water extracts and identified as a 41 kD glycopeptide. Treatment with 200 microg/mL LBGP3 increased the apoptotic rate of T cells from aged mice and showed a similar DNA ladder pattern to that in young T cells. The reversal of apoptotic resistance was involved in down-regulating the expression of Bcl-2 and FLIP, and up-regulating the expression of FasL. CONCLUSION: Lycium barbarum glycopeptide 3 reverses apoptotic resistance of aged T cells by modulating the expression of apoptosis-related molecules.
Assuntos
Envelhecimento/imunologia , Apoptose/efeitos dos fármacos , Glicopeptídeos/farmacologia , Lycium/química , Linfócitos T/efeitos dos fármacos , Animais , Proteína Ligante Fas/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , RNA Mensageiro/imunologia , Linfócitos T/imunologiaRESUMO
The mechanism of apoptosis induced by SIRT1 deacetylase inhibitors in both human breast cancer MCF-7 and MCF-7 doxorubicin-resistant cells was studied. MTT assay was used to detect growth-inhibitory effect on the cells. Protein expression was detected by Western blotting. Chromatin condensation was detected by a fluorescent microscope after Hoechst 33342 staining. Cell cycle distribution was analyzed with flow cytometry. Apoptotic cells were detected with Annexin V staining. Nicotinamide (NAM) and Sirtinol, two SIRT1 deacetylase inhibitors, exhibited the similar growth-inhibitory effects on MCF-7/DOX cells and MCF-7 cells, but no potentiation of DOX activities. The arrest at G2/M phase was detected by flow cytometry in both MCF-7 and MCF-7/DOX cells after NAM treatment. Activation of caspase pathway in MCF-7 cells, such as the cleavages of PARP, caspase-6, -7, -9, were observed after exposure to NAM 50 mmol x L(-1), accompanied by the occurrence of chromatin condensation and Annexin V positive cells. However, the cleavages of PARP, caspase-6 and -7 in MCF-7/DOX cells delayed after exposure to NAM for 24 h and obviously increased at 48 h with appearance of chromatin condensation and Annexin V positive cells. SIRT1 deacetylase inhibitors show no cross resistance to MCF-7 drug-resistant cells, and the similar growth-inhibitory actions of them to MCF-7 sensitive and drug-resistant cells by which it is mediated by activation of apoptotic caspase pathway.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Benzamidas/farmacologia , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Naftóis/farmacologia , Niacinamida/farmacologiaRESUMO
OBJECTIVE: To investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence. METHODS: Chromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-beta-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA. RESULTS: Mitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-beta-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells. CONCLUSION: Mitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Enedi-Inos/farmacologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Telomerase/metabolismo , Apoptose/efeitos dos fármacos , Corantes Azur , Benzimidazóis , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Cromatina/metabolismo , DNA de Neoplasias/análise , Relação Dose-Resposta a Droga , Genoma Humano/genética , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitose/efeitos dos fármacos , Fenótipo , Propídio , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
Enediyne compound is one class of antibiotics with very potent anti-cancer activity. However, the role of p53 in enediyne antibiotic-induced cell killing remains elusive. Here we reported the involvement of p53 signaling pathway in apoptosis induction by lidamycin (LDM), a member of the enediyne antibiotic family. We found that LDM at low drug concentration of 10 nmol/L induces apoptotic cell death much more effectively in human colorectal cancer cells with wild type p53 than those with mutant or deleted p53. p53 is functionally activated as an early event in response to low dose LDM that precedes the significant apoptosis induction. The primarily activation of mitochondria as well as the activation of p53 transcriptional targets such as Puma, Bad and Bax in HCT116 p53 wild type cells further demonstrates the key role of p53 in mediating the compound-induced apoptosis. This is further supported by the observation that the absence of Bax or Puma decreases apoptosis dramatically while Bcl-2 overexpression confers partially resistance after drug treatment. Activation of p53 signaling pathway leads to activation of caspases and caspases inhibitor VAD-fmk completely blocks low dose LDM induced apoptosis through the inhibition of mitochondria pathway. In contrast, LDM at higher concentration causes rapid apoptosis through more direct DNA damaging mechanism that is independent of activation of p53 and caspases and cannot be blocked by caspase inhibitor. Taken together, LDM induces apoptosis in a p53-dependent manner when given at low doses, but in a p53-independent manner when given at high doses. This dosage-dependent regimen can be applied to cancer clinic based upon the p53 status of cancer patients.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Enedi-Inos/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Relação Dose-Resposta a Droga , Humanos , Marcação In Situ das Extremidades Cortadas , Potenciais da Membrana , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio , Transdução de SinaisRESUMO
Although enediyne antibiotic lidamycin ( LDM) is a potent inducer of apoptosis, the underlying mechanisms of its apoptotic functions remain to be explored. Here, we aim to elucidate its possible mechanisms in mitochondria initiated apoptotic pathway involved in human BEL-7402 and MCF-7 cells. Cytochrome c released from mitchondria to cytosol fraction was detected by Western blotting. p53 and Bax, Bcl-2 expressions were detected by Western blotting and RT-PCR. MTT assay was used to detect cytotoxicity of LDM with or without caspase inhibitor z-VAD-fmk. After the BEL-7402 cells were exposed to 0. 1 micromol x L(-1) LDM within 6 h, the increase of cytochrome c in the cytosol and decrease in the mitochondria were observed when compared with untreated cells. The expression of Bax, an important proapoptotic member of the Bcl-2 family, increased gradually in the BEL-7402 cells after exposure to LDM of 0. 1 micromol x L (-1) for 2, 6, and 9 h, separately, while Bcl-2 increased at 2 and 6 h, and decreased at 9 h after LDM treatment. Enhanced protein expressions were parallel with respective increased mRNA level for Bax only, but not p53. Caspase inhibitor may inhibit partially the killing effects induced by LDM. Therefore we conclude that the rapid activation of mitochondrial pathway induced by LDM in tumor cells might contribute to its highly potent cytotoxicities.
Assuntos
Aminoglicosídeos/farmacologia , Apoptose/efeitos dos fármacos , Enedi-Inos/farmacologia , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Western Blotting , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
A new antibiotic, S632A3, was isolated from a cultured broth of Streptomyces hygroscopicus S632. It was purified by column chromatography on silica gel, Sephadex LH-20 and HPLC. Structural studies by analysis of 1H NMR and 13C NMR, MS, UV and IR spectra in comparison with those of S632A2 clarified that S632A3 is an isomer of 9-methylstreptimidone. In addition, this antibiotic showed potent biological activity including differentiation induction effects on HL-60 cell and antitumour activity in vivo.
Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Piperidonas/química , Piperidonas/farmacologia , Streptomyces/química , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Células HL-60 , Humanos , Camundongos , Estrutura Molecular , Piperidonas/isolamento & purificaçãoRESUMO
AIM: Mitotic cell death has been focused on in tumor therapy. However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the possible link between centrosome dynamics and lidamycin-induced mitotic cell death in human hepatoma BEL-7402 cells. METHODS: Growth curve was established by MTT assay. Cell multinucleation was detected by staining with Hoechst 33342. Flow cytometry was used to analyze cell cycle. Aberrant centrosomes were detected by indirect immunofluorescence. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively. RESULTS: Exposure of BEL-7402 cells to a low concentration of lidamycin resulted in an increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death and activation of SA-beta-gal in some cells, accompanied by the changes of protein expression for the regulation of proliferation and apoptosis. The mitochondrial signaling pathway, one of the major apoptotic pathways, was not activated during mitotic cell death. The aberrant centrosomes contributed to the multipolar mitotic spindles formation, which might lead to an unbalanced division of chromosomes and mitotic cell death characterized by the manifestation of multi- or micronucleated giant cells. Cell cycle analysis revealed that the lidamycin treatment provoked the retardation at G2/M phase, which might be involved in the centrosome overduplication. CONCLUSION: Mitotic cell death and senescence can be induced by treatment of BEL-7402 cells with a low concentration of lidamycin. Centrosome dysregulation may play a critical role in mitotic failure and ultimate cell death following exposure to intermediate dose of lidamycin.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Carcinoma Hepatocelular , Morte Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Neoplasias Hepáticas , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Enedi-Inos , Humanos , Mitocôndrias , Mitose/efeitos dos fármacos , Fenótipo , Poliploidia , Fuso Acromático/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate whether two kinds of in vitro prepared advanced glycation end products (AGEs), Glu-BSA and Gal-BSA, could change oxidation stress and anti-oxidation abilities in astrocytes, and thus might contribute to brain injury. METHODS: Changes of GSH, MDA, SOD, MAO-B, nitric oxide were measured after AGEs treatment. RESULTS: Both 0.1 g/L Glu-BSA and Gal-BSA could slightly decrease GSH level, while 1 g/L of them significantly decreased GSH level by 35% and 43% respectively. The MDA levels of both 1 g/L AGEs treated groups (306 +/- 13 and 346 +/- 22) were higher than that of the normal group (189 +/- 18), which could be inhibited by free radical scavenger NAC. The SOD activities of both 1 g/L AGEs treated groups (67.0 +/- 5.2 and 74.0 +/- 11.0) were lower than that of the normal group (85.2 +/- 8.0). Both 0.1 g/L AGEs could slightly increase the activity of MAO-B, while 1 g/L of them could increase MAO-B activity by 1.5 and 1.7 folds respectively. Both AGEs stimulation could produce NO level by 1.7 and 2 folds respectively. CONCLUSION: Enhanced levels of astrocytic oxidation stress and decrease of antioxidation abilities may contribute to, at least partially, the detrimental effects of AGEs in neuronal disorders and aging brain.
Assuntos
Astrócitos/efeitos dos fármacos , Produtos Finais de Glicação Avançada/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Soroalbumina Bovina/farmacologia , Animais , Astrócitos/enzimologia , Astrócitos/metabolismo , Bovinos , Células Cultivadas , Córtex Cerebral/citologia , Glutationa/metabolismo , Malondialdeído/metabolismo , Monoaminoxidase/metabolismo , Óxido Nítrico/metabolismo , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
AIM: To investigate the synergetic effect and the mechanism of antitumor action of the antibiotic lidamycin in combination with cisplatin in vitro. METHODS: Cytotoxicity of the drugs was measured by clonogenic assay. Chromatin condensation was observed by co-staining with fluorescent dyes, Hoechst 33342 and propidium iodide. Apoptotic sub-G1 was detected by flow cytometry and DNA ladder was observed using agarose gel electrophoresis. Bcl-2 protein level was detected by Western blot assay. RESULTS: By using clonogenic assay, lidamycin in combination with cisplatin was found to have synergetic effects on the proliferation of human hepatoma BEL-7402 cells. The data showed that BEL-7402 cells treated with cisplatin and lidamycin in combination produced internucleosomal DNA fragmentation analysed by agarose gel electrophoresis. The results of flow cytometry showed that cisplatin and lidamycin administrated in combination showed no obvious change in G1 phase distribution compared with single treatment. However, this combination reduced the S phase arrest and reversed the reduction of G2/M phase induced by single treatment. The results also showed that there was 11.3% or 9.37% of cells undergoing apoptosis in BEL-7402 cells treated with cisplatin or lidamycin, respectively, while it showed 32.4% of apoptotic cells in combination treatment. Cisplatin, lidamycin and combination of cisplatin and lidamycin was shown to induce typical chromatin condensation in BEL-7402 cells. The study showed that 0.5 mumol.L-1 cisplatin or 1 x 10(-4) mumol.L-1 lidamycin alone decreased Bcl-2 protein level, while lidamycin in combination with cisplatin strongly inhibited expression of Bcl-2 proteins in BEL-7402 cells. CONCLUSION: The results suggest that lidamycin enhancement of cisplatin-induced apoptosis associates with decrease of Bcl-2 protein expression, which may be useful for cancer chemotherapy.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose , Cisplatino/farmacologia , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/patologia , Sinergismo Farmacológico , Enedi-Inos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fase S/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Enediyne antibiotics have been reported to be the most potent cytotoxic antitumor agents. The pathway by which these compounds cleave DNA and induce apoptosis of tumor cells may be different from the caspase-mediated pathways that initiate typical apoptosis. In this report, we studied the apoptosis induced by lidamycin (LDM), a member of the enediyne antibiotic family, and compared the characteristics of LDM-induced apoptosis with those of typical apoptosis induced by mitomycin C or etoposide. Chromatin condensation occurred very rapidly and appeared as speckles in human hepatoma BEL-7402 and breast carcinoma MCF-7 cells after treatment with 1 microM LDM. In addition, co-staining the cells with the mitochondria-specific dye Mitosensor and the DNA-specific dye Hoechst 33342 enabled the visualization of mitochondria in normal control and LDM-treated cells but not in mitomycin C-treated cells. Neither the caspase inhibitor VAD-fmk nor the caspase-3 inhibitor DEVD-fmk was able to inhibit the DNA ladder patterns caused by LDM in BEL-7042 or MCF-7 cells. Smaller fragments of histone H1 cleaved by LDM were detected by SDS-PAGE, indicating that the site of LDM action is the internucleosomal structure. Although caspase-9, caspase-3/7, and caspase-6 activities were increased in BEL-7402 cells, and caspase-7 activity was increased in MCF-7 cells after treatment with 1 microM LDM, this occurred much later, indicating that chromatin condensation reached the maximal level rapidly while caspase activities still remained low. Taken together, these results demonstrate that LDM induced rapid DNA cleavage and chromatin condensation independently of caspase activities; this may contribute to its highly potent cytotoxicity toward tumor cells.
Assuntos
Aminoglicosídeos , Antibacterianos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose , Cromatina/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias da Mama/patologia , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Caspases/fisiologia , Sobrevivência Celular/efeitos dos fármacos , DNA/efeitos dos fármacos , Enedi-Inos , Ativação Enzimática , Histonas/metabolismo , Humanos , Mitocôndrias/metabolismo , Células Tumorais CultivadasRESUMO
AIM: To investigate whether two kinds of in vitro prepared advanced glycation end products (AGE), Glu-BSA and Gal-BSA, could induce proinflammatory mediators IL-1beta and TNF-alpha, as well as oxidative stress and nitric oxide (NO), in astrocytes, thus contributing to brain injury. METHODS: Radioimmunoassay and RT-PCR technique were used to detect two cytokines' level and existence of receptor for AGE (RAGE). DTNB reaction was used to measure reduced glutathione (GSH) level. NO content was assayed using Griess reagent provided by Promega. RESULTS: Enhanced protein levels of both cytokines in supernatants and cell lysates of astroglia cultures were detected after treated with AGE-BSA 1 g/L, especially Gal-BSA, for 72 h. The increases were also in a concentration-dependent manner. Changes in protein levels might be attributed to changes in transcriptional levels documented by semi-quantitative RT-PCR. Both AGE-BSA could also reduce astrocytic GSH and induce NO release. RAGE was detected in astrocytes. CONCLUSION: Enhanced levels of astrocytic proinflammatory mediators IL-1beta and TNF-alpha, and oxidative stress caused by AGE might contribute to, at least partially, the detrimental effects of AGE in neuronal disorders and aging brain.